4-Lower alkyl or aryl substituted-spiro{8 furan-2(3H),1{40 -phthalan{9 -3,3{40 -diones as fluorometric reagents

ABSTRACT

A novel class of reagents react with primary amine-containing compounds, preferably polypeptides and amino acids, to form highly fluorescent products. This reaction serves as a basis for a rapid and highly sensitive assay method for primary aminecontaining compounds.

' United States Patent [191 Leimgruber et al.

[[ Nov. 4, 1975 4-LOWER ALKYL OR ARYL SUBS'IITUTED-SPIRO[FURAN-2( 3H),1 PHTHALAN]-3,3-DIONES AS FLUOROMETRIC REAGENTS [75] Inventors: Willy Leimgruber, Montclair;

Manfred Weigele, North Caldwell,

both of NJ.

[73] Assignee: Hoffmann-La Roche Inc., Nutley,

[22] Filed: Mar. 4, 1974 [21] App]. No.: 447,711

Related US. Application Data 62] Division of Ser. No. 212,790, Dec. 27, 1971, Pat. No.

[52] US. CL... 260/326.l3 R; 260/3405; 260/3433 [51] Int. Cl. C07D 209/18; CO7D 307/88;

C07D 317/50 [58] Field of Search..;... 260/343.3, 340.5, 326.13 R

Primary ExaminerLewis Gotts Assistant ExaminerS. P. Williams Attorney, Agent, or Firm-Samuel L. Welt; Jon S. Saxe; Raymond R. Wittekind 19 Claims, No Drawings 4-LOWER ALKYL OR ARYL SUBSTITUTED-SPIRO[FURAN-2(3H),1 PHTHALAN]-3,3-DIONES AS FLUOROMETRIC REAGENTS 4-( Z-methoxyphenyl )spiro'[furan-2( 3 H), l -phthalan This is a division of application Ser. No. 212,790,

filed Dec. 27, 1971, now U.S. Pat. No. 3,812,181.

detection and assay of amino acids, amines and peptides has been known in the art for nearly 60 years. It

serves as the basis for the well known Stein-Moore procedure now extensively utilized in the automated assay of amino acids. McCaman and Robins described a fluorometric method for the detection of serum phenylalanine involving the reaction between ninhydrin and phenylalanine and discovered the fluorescence was greatly enhanced by the addition of a variety of peptides. Later, Udenfriend and coworkers, Anal. Biochem. 42, 222, 237 (1971), described a fluorometric assay procedure for primary amine-containing compounds, especially peptides and amino acids, which involved the reaction between the primary amine-contained compound, ninhydrin and an aldehyde, preferably phenylacetaldehyde. This procedure was shown to be much more efficient and sensitive than the Stein- Moore and McCaman-Robins procedures.

BRIEF DESCRIPTION OF THE INVENTION 'Thepresent invention relates to a novel series of compounds represented by formula I wherein R is hydrogen, halogen, lower alkyl or lower alkoxy and'R" is lower alkyl or aryl;

These compounds produce highly fluorescent sub- 1 stances upon reaction with primary amine-containing compounds, and are therefore designated as fluoroens. g In the specification and the appended claims, the term lower alkyl shall mean a monovalent, saturated, straight or branched chain hydrocarbon substituent containing up toand including 8 carbon atoms; the term lower alkoxy shall mean a group having a lower alkyl group linked to an ether oxygen and having its free valence bond from the ether oxygen; the term aryl shall mean an aromatic ring system which may be substituted with one or more of the following: halogen (i.e., fluorine, chlorine, bromine or iodine), lower alkyl, lower alkoxy, nitro, cyano and so forth. Exemplary aromatic ring systems include phenyl naphthyl, fury], thienyl, pyrrolyl, imidazolyl, pyridyl, pyrimidinyl,

vindolyl, quinolyl, oxazolyl, isoxazolyl, and so forth.

Preferred compounds of formula I are those where R is hydrogen. Examples of preferred compounds of formula I are: 4-phenylspiro[furan-2( 3H ),1 -phthalan -3,3 '-dione 3,3-dione 4-( 3-methoxyphenyl)spiro[furan-2( 3H),1 '-phthalan 3,3-dione 4-( 4-methoxyphenyl )spiro furan-2( 3H ,1 -phthalan 3,3'-dione 4-( 2,4dimethoxyphenyl)spiro[furan-2( 3H),l

phthalan]-3,3-dione I 4-( 2,5-climethoxyphenyl )spiro[furan-2( 3H), 1

phthalan ]-3 ,3 -dione 4-( 3 ,S-dimethoxyphenyl)spiro[furan-2( 3H),1

phthalan]-3 ,3 -dione 4-( 3 ,4,5-trimethoxyphenyl )spiro[furan -2( 3H),l

phthalan -3 ,3 -dione 4-( 2,4,5-trimethoxyphenyl)spiro[furan-2( 3H),l

phthalan ]-3 ,3 '-dione 4-( 3 ,4-rnethylenedioxyphenyl )spiro[furan-2( 3H),l

phthalan -3 ,3 -dione 4-( 3-chlorophenyl )spiro [furan-2( 3H),l -phthalan]- 3,3-dione 4-(4-chlorophenyl )spiro [furan-2( 3H) l '-phthalan 3,3'-dione 4-(4-broinopher1yl)spiro[furan-2( 3H) l -phthalan'] dione 4-( l-naphthyl)spiro[furan-2(3H),l '-phthalan]-3,3

dione I 4-( 1-propyl)spiro[furan-2( 3H), 1 '-phthalari]-3,3

dione. Especially preferred is the compound-wherein R is phenyl, i.e. 4-phenylspiro [furan-2( 3H),1 "-pht'halanl- 3,3'-dione. i

The preparation of compounds of formula I is illustrated in Reaction Scheme A starting from'compound II. Compounds of formula II are generally known and are prepared starting with the properly substituted 0- acetyl benzoic acid and the desired aldehyde R'CHO. Where particular members of the genus represented by formula II have not been previously described in the art, they may be prepared in the same manner as the known ones. n

In the first step, the enol lactone of formula II is hydrolyzed to the keto carboxylic acid ofyformula III by an aqueous basic hydrolysis, followed by acidification. Suitable bases for the hydrolysis reaction include alkali metal hydroxides, e.g. sodium hydroxide, alkali metal carbonates, e.g. sodium carbonate, and alkali metal bicarbonates, e.g. sodium bicarbonate. A preferred base is an alkali metal hydroxide such as sodium hydroxide. The temperature of the hydrolysis can be in a range of from about 0 to about C. A preferred temperature range is from about 10 to about 40C. The hydrolysis reaction is suitably performed. in an aqueous medium. An organic co-solvent such as a lower alkanol or an organic ether may be employed if desired. At the completion of the reaction, the reaction mixture is acidified to free the carboxylic acid reaction product for the purposes of isolation.

Compounds of formula III are depicted as existing in both the diketo and the enolized form. It should be understood that the ratio of tautomers of the compound can vary depending upon solvent, temperature, pH, and so forth. In the experimental section and the claims, the compounds of formula III are named as the enol form for convenience only.

REACTION SCHEME A R l I wherein R and R are above; R and R taken independently are each lower alkyl; and R and R taken together with the nitrogen atom form a 5- or 6-membered saturated heterocyclic ring having at the most one additional heteroatom selected from the group consisting of nitrogen and oxygen.

The keto carboxylic acid of formula III may be converted to the fluorogen of formula I by a two step procedure. In the first step the keto acid of formula III is reacted with an amino-methenylating agent to afford an enamine of formula IV. Suitable aminomethenylating agents include acetals of an N,N-disubstituted fonnamide, e.g. dimethylformamide dimethylacetal; tris(secondary amino)methanes, e.g. tris(dimethylamino)methane and tris(piperidino) methane; and bis(secondary amino)lower alkoxy methanes, e.g. bis (dimethylamino)-t-butoxy methane.

The amino moiety as shown in the structural formula for compound IV is introduced from the aminomethenylating agent. Acetals of N,N-disubstituted formamides have the general formula,

tris(secondary amino)methanes have the general formula H-C N and bis(secondary amino)lower alkoxymethanes have the general formula Examples of amino moieties include those where R and R each taken independently are lower alkyl, e. g. dimethylamino and diethylamino; and those where R and R taken together with the nitrogen form a 5- or 6-membered heterocyclic ring, e.g. piperidino, morpholino, pyrrolidino, and so forth. I

This reaction may be carried out in any inert organic solvent. Particularly preferred solvents include formamides, especially dimethylformamide. An excess of aminomethenylating agent may be also utilized as solvent. The preparation of the enamine may be effectuated over a temperature range of from about 0 to about although a temperature range of from about 10 to about 40 is preferred. A temperature of about room temperature is especially preferred.

The enamine of formula IV may be then directly converted to the fluorogen of formula I by aqueous hydrolysis at a neutral acidic or basic pH. Thus, an acid such as a mineral acid, e.g. I-ICl; or a weak base such as an alkali metal bicarbonate, e.g. sodium bicarbonate, may be present in the hydrolysis medium. In general, it is preferred to effectuate the aforementioned hydrolysis in the presence of a weak base such as an alkali metal bicarbonate. Upon completion of the hydrolysis and acidification of the reaction medium (in cases where the hydrolysis is carried out on the neutral or basic side) the desired cyclized fluorogen of formula I is obtained. The temperature of the hydrolysis reaction is suitably in the range of from about 0 to about 100, although a temperature of about 10 to 40 is preferred. A temperature of about room temperature is especially preferred. The conversions II- III, III+IV and IV- I may be carried out with isolation and purification of the product at each stage, or preferably, can be carried out with isolation and purification only of the final fluorogen of formula I.

The fluorogens of formula I react readily with primary amine-containing compounds, particularly with peptides, amino acids and biogenic amines, e.g. catecholamines, to form highly fluorescent materials. Thus, the compounds of formula I serve as highly sensitive reagents for the detection of primary amine-containing compounds. Since the compounds of formula I are stable .to aqueous media, the detection of primary aminecontaining compounds present in said media isrendered highly practical. This is especially important for the detection of amino acids and peptides which are generally found and analyzed in aqueous media.

The reaction between fluorogens of formula I and primary amine-containing compounds is a rapid and quantitative one. The reaction may be carried out over a wide pH range of from about pH 4 to about pH 11. However, the rate and completeness of the reaction of the fluorogen with the primary amine-containing compounds is optimal within a pH range between about 8 and 9. Thus, reaction of a compound of formula I with a simple primary amine, such as ethylamine, is instantaneously complete at room temperature within this pH range.

It is immediately apparent that the use of a fluorogenic reagent of formula I for the detection of primary amine-containing compounds is superior to the prior art technique hereinabove described involving a ternary reaction between a primary amine-containing compound, ninhydrin and phenylacetaldehyde. A further superiority of the reagents and methods of the present invention is seen from the fact that, inthe prior art procedure, it was necessary to heat the mixture of reagents to a temperature of about 60C. for 15 to 30 minutes to develop maximum fluorescence, whereas, in contradistinction, the reagent of the present invention develops maximum fluorescence instantaneously at room temperature. The fluorescence obtained using equal concentrations of primary amine-containing compounds utilizing the reagents of the present invention is generally in the range of 10 to 50 times greater than that obtained by using the prior art technique. Thus, the reagents of thepresent invention may be utilized to detect exceedingly minute quantities of primary amine-containing compounds which was heretofore impossible. Due to the sensitivity of the reagents of the present invention, small, amounts of peptides and amino acids can be detected. For example, one can detect and examine quantities of biologicallyactive peptides isolated from a given organ or a single laboratory animal.

The use of the reagents of the present invention for the detection of primary amine-containing compounds may be illustrated in a number of ways. For example, the presence of primary amine-containing compounds in solution may be determined by mixing the sample with an excess of fluorogen of formula I at a desired pH, and measuring the fluorescence. It is preferred to have the primary amine-containing compound in solution in an aqueous medium and to treat it with a solution of the fluorogen in a non-hydroxylic, water miscible solvent such as acetonitrile, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dioxane; and the like. A l0-fo1d to 5000-fold molar excess of fluorogen is preferred. A 50-fold to 1 OOO-fold molar excess is especially preferred.

Alternatively, the fluoro gens of the present invention may be utilized to detect the presence of primary amine-containing compounds, especially amino acids, on paper or thin-layer chromatographic systems. In such a technique, the reagent in a solvent, preferably a ally as a spray, and the paper or thin-layer chromatogram is examined under a fluorescent light source. The development of fluorescence is instantaneous at room temperature and, thus, there is no need to heat the chromatogram to develop fluorescence. This offers a decided advantage over prior art techniques.

Another application of the fluorogens of the present invention is the use in automated analysis of amino acids. In such a procedure, a sample stream which is a portion of the effluent of a chromatographic column is mixed with the fluorogenic reagent of formula I at the desired pH and the mixture immediately analyzed by a spectrofluorometer. In this manner, a monitoring of the fluorescence exhibited by a continuous sample stream is indicative of the presence and concentration of various amino acids.

A further appreciation of the preparation of the novel fluorogens of the present invention andtheir use in producing highly fluorescent substances upon reaction with primary amine-containing compounds may be obtained from the following examples.

EXAMPLE 1 A solution of I 8.1 g., l,4-isochromandione (0.05 mole), 6.0 g. benzaldehyde (0.055 mole) and 0.1 ml. piperidine in. 50 ml. benzenewas heated at reflux temperature under nitrogen until the theoretical amount of water (0.05 mole) had collected in a Dean-Stark trap (ca. 2 .hrs.).-The reaction mixture was then cooled to 10 and the product was filtered off. The filter cake was recrystallized from benzeneto afford 1 1.4 g., 3-benzylidene-4-keto-3,4-dihydroisocoumarin, m.p. 177.

Anal. Calcd. for C H O C, 76.79; H, 4.03 Found: C, 76.49; H, 4.05.

Analogously were prepared from l,4-chromandione and thecorresponding aldehydes the compounds listed in Table I: a

Table I s Empirical Analysis R'= mp Formula Calcd. Found 2-methoxyphenyl 200 C H Q, C, 72.85 72.96 H, 4.32 4.19 3-methoxyphenyl 153 C H O, C, 72.85 72.52 H, 4.32 4.21 4-methoxyphenyl 200 C H Q, C, 72.85 72.56 H, 4.32 4.29 2,4-dimethoxyphenyl 201 C H O C, 69.67 69.53 H, 4.55 4.57 2,5-dimeth0xyphenyl C ,H,.,O C, 69.67 69.65 H, 4.55 4.54 3,5-dimethoxyphenyl 178 C, H,,O C, 69.67 69.62 I H, 4.55 4.63 3,4,5-trimethoxyphenyl 144 C H Q, C, 67.05 66.71 H, 4.75 4.56 2,4,5-trimethoxyphenyl 210 C H O C, 67.05 67.35 H, 4.75 4.77 3,4-methylenedioxyphenyl 226 C I-1, 0,, C, 69.39 69.16 H, 3.43 3.33 3-chlorophenyl 224 C H CIO C, 67.50 67.41 H, 3.19 3.21

continued -cntinued Empirical Analysis Empirical Anal is R= mp Formula Calcd. Found R= mp Formula Calcd. Found 4 6h16id heh 1 203 10,11,00 c, 67.50 67.26 1 H. 4.17 4.06 r H, 3.19 3.04 2,5-dimethoxyphenyl 148 CWHHOS C, 67.45 67.54 4-bromophenyl 204 C'WHQBJ'OQ C 58.38 58.46 H, 4.17 4.09 5 H, 2.76 2.66 3",5-dimethoxyphenyl 138 CWHHOG C. 67.45 67.72 3.ihd 61 1 308 C ,..H..N.0, c, 74.73 74.69 H, 4.17 3.98 H, 3L83. 3,94 3,4,5-trimethoxyphenyl 205 CZUHWOQ C, 65.21 65.33 I N, 4.84 4.92 H, 4.38 4.26 Z-naphthyl '25-1 CQDHHOQ 7C, 79.99 80.28 10 -2,4,5-trimethoxyphenyl 179 CZUHISO1 C, 65.21 -65.34 1-1, 4.03 4.01 1-1, 4.38 4.27 l-riaphthyl 184 c ii o c, 79.99 80.27 3,4 methylenedioxyphenyl 195 0811.00.i C. 67.08 67.08 I 1 1 1-1, 4.03 3.95 H. 313 3.43 n propyl 58" C,3H,2Q3 C, 72.21 72.01 3-chlorophenyl 144 CWH ClO C, 65.30 65.32 H, 5.59 5.44 1-1, 2.90 2.61 '7 4-chlorophenyl 167 CHHQCIQ, C, 65.30 65.10 H, 2.90 2.92 l5 4-bromophenyl 181 C l-l BrO C, 57.17 57.25 a H, 2.54 2.63 a EXAMPLE 2 3-ihd61 1 186 c l-l No, c, 71.92 72.16 1 I H, 3.49 3.43 To a solution of g. 3-benzyl1dene-4-keto-3,4-d1hy- N, 4.41 4.16 droisocoumarin in 400 ml. methanol were added 100 zmaphthyl (321131204 2g: 3 23 aqueous sodium hydroxide. The mixture was 20 lmapmhyl 7 CMHHO, 3 3; 76 35 stlrred at room temperature for 30 mmutes. It was then C H o H. 3.68 3.53 diluted with water and acidified with 10% hydl'Ochlolic Y H 1 'acid. The precipitating o-(a-hydroxycinnamoyl) benzoic acid was extracted twice with chloroform. The organic extracts were washed with water, dried over so- 25 EXAMPLE 3 dium sulfate and evaporated under reduced pressure to dryness. The residue was dissolved in 80 ml. dimethyl- T odaflsuspenslon. f i' g f s ffigg gfd formamide. The solution was cooled to 0, and, while y gY a g fi g 3 h i k f z was stirring, 24 ml. tris(dimethylamino)methane were t queot S o h A d k d added. Stirring was continued at room temperature for f a i g 5 or g j d f re 60 minutes; The l-dimethylamino-2-phenyl-4(o-car- 3;. g g 0 1 8 3233? 'boxyphenyl)-1 buten-3,4-dione which had formed was a" t 5 9 13 if -not isolated, but rather, the DMF-solution was poured e W1 g 3 e into ice-water. The resultant aqueous alkaline solution 5 3 5 ex rac f g was e i wa was extracted with ether. The ether extract was'dis- 5 33 82 2 g g p i g carded and the aqueous layer was acidified with dilute t etresl ue was 3 m y em d hydrochloric acid. The desired 4-phenylspiro[furann :h r 1 ffi fP P f e g 3 a T1 1 2( 3'H)-1-ph'thalan]3,3-dione was extracted three times to so to e 01m 0 mclplent tur l 2 with ether/benzene (1:1); The combined extracts were L g g E ywfwgfg or al dried over sodium sulfate and evaporated'under re- 40 g T; al a if)? duced pressure. The crystalline residue was resuslze an tere o e cryt s were was e on pended in ether and filtered off to give 12.0 G. pure the filter wlt,h methylene chlonde/petroleum ether material mp 6 (1:9) and dried in vacuo to afford 2.0 g. of o-(a- 9 c 0 Anal. Calcd. for 0, 11, 0 c, 73,38; H, 3.62 Found: hYdYOXYCmHamOYDPeHZQIC aclda P 99 C 73 H 3 57 45 The compounds listed in Table III were prepared by 7 7 9 n a The compounds listed in Table II were prepared by analiogous proceduresastamqg appropnate analogous procedures, starting with the appropriate 3- F ldene'l4'keto3Adlhydrolsocoumarms, Prepared as arylidene- OI 3-alkylidene-4-keto-3,4-dihy- EXamP e droisocoumarins, prepared as in Example 1.

g 5 Table III Table II 2 Empirical Analysis H R= mp Formula Calcd. Found 0 0 2 -methoxyphenyl l3l136 CHHHOS C, 68.45 68.14 c, 4.73 4.73 3-methoxyphenyl 133l35 CHHHO5 C, 68.45 68.75 Empirical Analysis H, 4.73 5.00 R= mp p m Ca|cd Found 4-methoxyphenyl 117-123 CHHHOS C, 68.45 68.52 11, 4.73 4.78 2-methoxyphenyl 153 C,,,H|2O5 g, 2,5-dimethoxyphenyl l3ll32 C H o C, 65.85 65.82 H, 4.91 5.20 3-methoxyphenyl 118 C H O g, 7?.53 72.113 3,5-dimethoxyphenyl 13ll34 CWHSOB C, 65.85 66.08 2 1 H, 4.91 5.03 4-methoxyphenyl 158 CWHHOS g, 72.80 2,4,5-trimethoxyphenyl l52-l65 C H O C, 63.68 63.33 1 H, 5.06 5.11 2,4-dimethoxyphenyl 133 C|9H|4O8 C, 6745 67.21 3-chlorophenyl 1l7-l 18 C H clO C, 63.48 63.59

-continued l Empirical Analysis R'= mp Formula Calcd. Found I H, 3.66 3.63 4-hrom ophenyl l30-l32" C H BrO C, 55.35 55.37 i v H, 3.19 3.24 3-indolyl l20-l25 C H NO C, 63.52 63.63 v H, 4.03 3.78 v N. 4.01 3.94 l-naphthyl l54l60 C H O C. 75.46 75.53 H, 4.43 4.61

EXAMPLE 4 To a solutionof 268 mg. o-(a-hydroxycinnamoyD- be'nzoic acid in ml. of dimethylformamide were added 2 ml. dimethylformamide dimethyl acetal. The reaction mixture was kept at room temperature for 17 hours. The solution containing l-dimethylamino-Z- phenyl-4(o-carboxyphenyl l -butene-3,4-dione was then poured into water, acidified with dilute hydrochloric acid andextracted with methylene chloride. The organic extract was dried over sodium sulfate and concentrated in vacuo. The concentrate was applied to a short silica gel column (4.5 g.) and eluted with methylene chloride. The eluate was evaporated in vacuo and the residue was was recrystallized from methylene chloride/ether. There were obtained 1 mg. 4-pheny1- spiro[furan-2( 3H ),1 '-phthalan]3,3 -dione, mp

EXAMPLE 5 I and evaporated invacuo. The residue containing o-(ahydroxycinnar noyUbenzoic acid was dissolved in 150 ml. dimethylformamide. To this solution were added 30 g. tris-(piperidino)methane. The mxiture was stirred at room temperature for 5 hours and the solution containing 1-( 1-piperidino)-2-phenyl-4-(o-carboxyphenyl)lbuten-3,4-dione then poured'into ice-water. The alkaline aqueous'mixture was extracted with ether. The ether extract was discarded. The aqueous layer was acidified with 1N hydrochloric acid (300 ml.) and extracted four times with methylene chloride. The organic extracts were combined, washed with water, dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue was dissolved in 75 m1. methylene chloride and treated with l g. of Norite at reflux temperature. The hot solution was filtered. The filtrate was concentrated to 50 ml. and then diluted with d ml. ether. From the mixture there crystallized 8.4 g. of 4-phenylspiro[furan-2(3H),1 -phthalan]- 3,3-dione; mp ll53.

The compounds listed in Table II were prepared by analogous procedures, starting with the appropriate 3- arylideneor 3-alkylidene-4-keto-3,4-dihydroisocoumarins prepared as in Example 1.

EXAMPLE 6 To a solution of 4.47 g. o-(a-hydroxy-2-methoxycinnamoyl) benzoic acid in 30 ml. dimethylformamide 1(ll were added 5g. tris-(dimethylamino)-methane. The mixture was stirred at room temperature for 3% hours. Most of the excess formylating agent and the solvent were then removed under reduced pressure at 5560. The residue containing l=dimethylamino-2-(o-methoxyphenyl)-4-(o-carboxyphenyl)-1-bute n-3,4-dione V was redissolved in 10 ml. of dimethylformamide and this solution added to 100ml. water. The resulting alkaline mixture was extracted with methylene chloride. The

organic extract was discarded. The aqueous layer was acidified with dilute hydrochloric acid and extracted twice with methylene chloride. .The organic phases were combined, washed with water, dried over sodium sulfate and evaporated to dryness under reduced pressure. The remaining oil was dissolved in ether. Crystals precipitated from the ether solution upon refrigeration. They were collected by filtrationto afford 2.4 g. 4-(2- methoxyphenyl) spiro[furan-2( 3H),l '-phthalan].-3,3 dione, mp 152l54.

The compounds listed in Table III were converted to the corresponding spirolactoneslisted in Table II by analogous procedures.

EXAMPLE 7 Fluorometric Assay Procedure:

Place in a test tube:

a. 1 ml. of peptide solution (20 nanomole leucyl-alanine in 1 ml. pH 8-phospl1ate buffer) and b. 2 ml. pH 8 buffer solution (Fisher). Whileagitating with a vibro-mixer add rapidly:

c. 1 ml. of reagent solution (containing 0.2 millimole of spirolactone of Table Ill in 100 ml. acetonitrile).

Final concentrations:

Leu-ala: 5 nanomole/ml reagent: 0.5 ,umole/ml (100 fold excess) acetonitrile: 25%

aqueous buffer: 75%

Measure fluorescence immediately.

Instrumentation: Farrand Spectrofluorometer Excitation: 390 nm; slits 10,10

Emission: 385 nm; slits 20,20'

Sensitivity 1 Results:

With 4-phenylspiro [furan-2( 3H), 1 '-phthalari]3 ,3 dione as the reagent a fluorescent intensity of 80, relative to a fluorescence intensity of (at the same excitation and emission settings) for a quinine standard, 10 mg/ml 0.01N H SO was observed.

The above experiment was repeated using ethylamine in place of leucylalanine, and ethanol in place of acetonitrile. The fluorescence intensity obtained by utilizing various spirolactone reagents of Table II is shown in Table IV. The fluorescence intensity obtained using 4-phenylspiro[furan-2( 3H),l -phthalan]3 ,3 '-dione has been arbitrarily assigned the value 100, and the other intensities are given relative to it.

Table IV-continu ed I Rel. Compound of Table [I A Fluorescence R= EX m Intensity 3,4,5r-trimethoxyphenyl I 390 475 60 n-propyl 370 445 47 .EXAMPLE 3 droisocoumarin,

2-(a-hydroxycinnamoyl)-5-methyl-benzoic acid, l-dimethylamino-2-phenyl-4(2-carboxy-4-methoxyphenyl l-buten-3 ,4-dione, '-methoxy-4-phenylspiro[furan-2( 3H), 1 '-phthalan 3,3-dione, 3-benzylidene-4-keto-7-chloro-3,4-dihydroisocoumarm, Z-(a-hydroxycinnatnoyD-S-chloro-benzoic acid, l-dimethylamino-2-phenyl-4(2-carboxy-4-chlorophenyl l -buten-3 ,4-dione, 5 '-chloro-4-phenylspiro[fu ran-2( 3H ,1 '-phthalan 3,3'-dione. We claim: 1. A compound of the formula 5 pyrrolyl, imidazolyl, pyridyl, pyrimidinyl, indolyl, quinolyl, oxazolyl and isoxazolyl, which radical may be unsubstituted or substituted with one or more halogen, lower alkyl, lower alkoxy, nitro or cyano groups.

2. The compound of claim 1 wherein R is hydrogen.

3. The compound of claim 2 which is 4- phenylspiro[- furan-2( 3H),l -phthalan]-3,3 -dione.

4. The compound of claim 2 which is 4-(2-methoxyphenyl)spiro[furan-2( 3 H), l -phthalan]-3 ,3 '-dione.

5. The compound of claim 2 which is 4-(3-methoxyphenyl)spiro[furan-2(3H),1 -phthalan]3 ,3 -dione.

6. The compound of claim 2 which is 4-(4-methoxyphenyl)spiro[furan-2( 3 H), l -phthalan ]-3,3 '-dione.

7. The compound of claim 2 which is 4-(2,4-dimethoxyphenyl)spiro[furan-2( 3H)-1 '-phthalan]-3 ,3 dione.

8. The compound of claim 2 which is 4-(2,5-dimethoxyphenyl)spiro[furan-2(3H),1 -phthalan]-3,3 dione.

9. The compound of claim 2 which is 4-(3,5-dimethoxyphenyl)spiro[furan-2( 3H),l -phthalan]-3,3 dione.

10. The compound of claim 2 which is 4-(3,4,5- trimethoxyphenyl)spiro [furan-2( 3H), 1 -phthalan]- 3,3'-dione.

11. The compound of claim 2 which is 4-(2,4,5- trimethoxyphenyl )spiro ;8 furan-2( 3H), 1 -phthalan]- 3,3'-dione.

12. The compound of claim 2 which is 4-(3,4- methylenedioxyphenyl)spiro[furan-2( 3H),l phthalan -3 ,3 '-dione.

13. The compound of claim 2- which is 4-( 3-chlorophenyl )spiro[furan-2( 3 H ,1 -phthalan ]-3 ,3 -dione.

14. The compound of claim 2 which is 4-(4-chlorophenyl)spiro-[furan-2( 3 H),l '-phthalan]-3,3 '-dione.

15. The compound of claim 2 which is 4-(4-bromophenyl)spiro[furan-2(3H),1 '-phthalan]-3,3 '-dione.

16. The compound of claim 2 which is 4-( 3-indolyl)- spiro[furan-2( 3H),l -phthalan]-3,3 '-dione.

17. The compound of claim 2 which is 4-(2-naphthyl)spiro [furan-2(3H) ,1 '-phthalan]-3,3 '-dione.

18. The compound of claim 2 which is 4- (lnaphthyl)spiro[furan-2( 3H) ,1 -phthalan]-3,3 -dione.

19. The compound of claim 2 which is 4-(1-propyl)- spiro[furan-2( 3H),l '-phthalan]-3,3 '-dione.

. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CQRRECTION PATENT N0. 2 3,917,646

DATED November 4, 1975 INVENTOR(S) Willy Leimgruber and Manfred Weigele It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 12, claim 11, line 31, 8 furan-Z" should be [furan-Z.

Column 12, claim 14, line 39, delete between spit-o and [furan.

Column 12, claim 18, line 46, "4- (lnaph" should be l (lna h-.

Signed and Scaled this Third Day of June 1980 [SEAL] Attest:

SIDNEY A. DIAMOND Arresting Ojficer Commissioner of Patents and Trademarks 

1. A COMPOUND OF THE FORMULA
 2. The compound of claim 1 wherein R is hydrogen.
 3. The compound of claim 2 which is 4-phenylspiro(furan-2(3H), 1''-phthalan)-3,3''-dione.
 4. The compound of claim 2 which is 4-(2-methoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 5. The compound of claim 2 which is 4-(3-methoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 6. The compound of claim 2 which is 4-(4-methoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 7. The compound of claim 2 which is 4-(2,4-dimethoxyphenyl)spiro(furan-2(3H)-1''-phthalan)-3,3''-dione.
 8. The compound of claim 2 which is 4-(2,5-dimethoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 9. The compound of claim 2 which is 4-(3,5-dimethoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 10. The compound of claim 2 which is 4-(3,4,5-trimethoxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 11. The compound of claim 2 which is 4-(2,4,5-trimethoxyphenyl)spiro;8 furan-2(3H),1''-phthalan)-3,3''-dione.
 12. The compound of claim 2 which is 4-(3,4-methylenedioxyphenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 13. The compound of claim 2 which is 4-(3-chlorophenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 14. The compound of claim 2 which is 4-(4-chlorophenyl)spiro-(furan-2(3H),1''-phthalan)-3,3''-dione.
 15. The compound of claim 2 which is 4-(4-bromophenyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 16. The compound of claim 2 which is 4-(3-indolyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 17. The compound of claim 2 which is 4-(2-naphthyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione.
 18. The compound of claim 2 which is 4- (1naphthyl)spiro(furan-2(3H),1''-phtHalan)-3,3''-dione.
 19. The compound of claim 2 which is 4-(1-propyl)spiro(furan-2(3H),1''-phthalan)-3,3''-dione. 